Eltroxin and Hesperidin mitigate testicular and renal damage in hypothyroid rats: amelioration of oxidative stress through PPARγ and Nrf2/HO-1 signaling pathway

Background Thyroid hormones (THs) regulate growth, development and function of different tissues. Hypothyroidism is a common clinical disorder characterized by deficiency in THs and adversely affects the development and functions of several organs. This work aimed to investigate the ameliorative effect of eltroxin (ELT), a hypothyroidism medication, and hesperidin (HSP), a flavonoid, against testicular and renal toxicity in hypothyroid rats. Twenty-four rats were divided into four groups and treated orally for 12 weeks. Group I (control), group II (hypothyroidism) received 20 mg/kg carbimazole (CBZ), group III received CBZ and 0.045 mg/kg ELT, and group IV received CBZ and 200 mg/kg HSP. Results CBZ administration induced biochemical and histopathological changes in testis and kidney. Co-administration of ELT or HSP significantly (P < 0.05) ameliorated THs, reduced urea and creatinine while raised follicle stimulating hormone (FSH), Luteinizing hormone (LH), and testosterone in serum. Testicular and renal malondialdehyde level as a lipid peroxidation indicator, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were significantly (P < 0.05) decreased while glutathione content, glutathione peroxidase, and glutathione-s-transferase activities were significantly (P < 0.05) increased. The histopathological changes were also diminished. Decreased mRNA and protein expressions of nuclear factor erythroid 2–related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and peroxisome proliferator-activated receptor gamma(PPARγ) in hypothyroid rats were up-regulated after ELT or HSP treatment. Conclusions ELT and HSP showed antioxidant and anti-inflammatory effects against CBZ-induced testicular and renal toxicity, and these effects may be promoted via activating Nrf2/HO-1 and PPARγ signaling pathways.


Background
Hypothyroidism (HPO) is a chronic disorder that affects about 10% of the worldwide individuals.It is characterized by a thyroid hormones (THs) insufficient supply of thyroxine (T4) and triiodothyronine (T3), which are important for regulating several metabolic processes, growth and development [1].Thyroid dysfunction should be properly and promptly treated due to the broad spectrum effects of THs on the body tissues and metabolism [2].Over time, untreated HPO may result in a number of health problems such as infertility, obesity, joint pain, and heart diseases [3].According to data from a prior research, low thyroid hormone levels may contribute to an increase in oxidative stress (OS) [4].
THs affect renal morphology and function since they are important for the growth and development.THs deficiency adversely affects the kidney function through altering intra-renal hemodynamics, and renin angiotensin aldosterone system (RAAS), as well as the structural changes including low kidney/body weight ratio, and abnormal glomerular architecture [5].Similarly, THs regulate some functions in the testis such as sperm motility, non-germ cells′ proliferation and differentiation, steroidogenesis, and testicular antioxidant status.Consequently, declined or raised THs can affect testicular function by different ways [6].
Eltroxin (ELT) (Levothyroxine) is a synthetic form of the thyroid hormone; T4 [7].Levothyroxine is the cornerstone of HPO therapy and is listed as one of the fundamental healthcare essential medications by the World Health Organization [3].This synthetic drug elevates serum T3 and T4 levels to regulate the thyroid gland activity [8].Prolonged use of a synthetic drug can treat HPO, but it may have adverse effects [9].Therefore, researchers are interested in flavonoids because of their abundance in food, possible health benefits, and relatively low toxicity when compared to other phytochemicals [10].Hesperidin (HSP), a pharmacologically active flavonoid aglycone, is widely distributed in citrus species (orange, blood orange, lime and lemon) [11].HSP was reported to have a broad variety of pharmacological effects including anti-thyroid, anti-inflammatory, antioxidant, anti-allergic, anti-cancer, anti-viral, anti-microbial, neuroprotective, and cardioprotective properties.It has been shown in several preclinical investigations that it protects against malignant transformation and development by acting on a variety of cellular signaling pathways [12,13].The antioxidant activities of HSP include increasing of cellular antioxidant defenses, quenching of free radicals, suppression of reactive oxygen species (ROS) producing enzymes, preventing DNA deterioration [14,15], and Nrf2 activation [16].Li et al. [17] has also verified that HSP exhibited a good safety profile when used in animal studies.
Nrf2 is a strategic antioxidant player in the thyroid gland [18].It has been demonstrated that Nrf2 has a role in the thyroidal function, it affects the basal and the thyroid-stimulating hormone (TSH)-induced boost of thyroglobulin, which is a key protein made by the gland and considers the precursor of T3 and T4 [19].Also, Nrf2 controls the expression of several anti-oxidant and cytoprotective genes in addition to tissue-specific genes required for the unique functional and homeostatic systems of each specific tissue in different organs [20].
Peroxisome proliferator-activated receptors (PPARs), ligand-inducible transcription factors and members of the nuclear receptor family, have a crucial role in maintaining the cells' regular activity.When PPARs are turned on, they heterodimerize with the retinoid X receptor (RXR), which in turn regulates the expression of several genes [21].Three PPAR isoforms have been identified; PPARα, PPARβ/δ, and PPARγ [22].PPARγ has an important role in male reproduction through regulating the genes for fatty acid metabolism [23].Also, PPARγ affects kidney development, and lipid metabolism [24].According to several reports, PPARγ and Nrf2 coactivation was proved to protect against inflammation and OS [25,26].
Consequently, we aimed to assess the protective effects of ELT and HSP against alterations in kidneys and testes provoked by CBZ-induced HPO in adult male rats, and to suggest the probable mechanism of action through testing the ability to modulate OS and inflammation possibly by activating PPARγ and Nrf2.

Chemicals
CBZ was purchased as tablets from Chemical Industries Development, Giza, Egypt.HSP was shipped from Sigma-Aldrich (St. Louis, MO, USA), and was stored away from sunlight.ELT was purchased from Glaxo Smith Kline GmbH, Germany.Other chemicals were of analytical grade and were obtained from commercial suppliers.

Experimental animals and treatments
In the current study, twenty-four adult male albino rats (Rattus norvegicus) weighing 130-150 g were purchased from the unit of animal housing in Nahda University (Egypt).The animals were kept in suitable well aerated cages at normal 12 h light/dark cycles, normal humidity (50-60%) and temperature (25 ± 3 °C) with free access to standard food pellets of known composition and water.Before the onset of the experiment, rats were observed for about 14 days to eliminate any possible infection.All animal treatments were accepted by the Institutional Research Ethics Committee of Beni-Suef University, Egypt (BSU-IACUC, No. 020-123).After the period of acclimatization, the rats were allocated into four equivalent groups with each group comprising six rats (N = 6), and were treated for 12 weeks.
Group I (CON): Orally gavaged with distilled water for one week, then 1% carboxymethyl cellulose (CMC) for 11 weeks.
All other rats received 20 mg/kg CBZ, dissolved in distilled water, through gastric intubation once/day for one week to induce HPO [27], then were separated into 3 groups and continued for 11 weeks as follows: Group II (CBZ): Rats received CBZ only throughout the experimental period.
Group III (CBZ + ELT): In addition to CBZ, rats were treated orally with 0.045 mg/kg/day ELT dissolved in distilled water [28].

Blood collection and tissue sampling
Twenty four hours following the determined experimental period, the animals from all groups were anesthetized intraperitoneally by 80 mg/kg ketamine/10 mg/ kg xylazine [30], and blood samples were collected from the medial canthus then rats were sacrificed by cervical dislocation.A median abdominal incision was achieved to expose the kidneys and testes which were carefully excised and washed with cold saline.
Serum was separated from blood samples after complete clotting, at room temperature, and centrifugation at 3000 rpm for 15 min.Serum was used for the analysis of testis and kidney function biomarkers and THs.
Samples from the testes and kidneys were homogenized (10% w/v) in cold phosphate buffered saline, centrifuged at 3000 rpm for 10 min, then the clear supernatant was separated and stored at -20 °C for the analysis of lipid peroxidation, antioxidants, TNF-α and IL-6.Other testis and kidney samples were fixed in Bouin's solution and 10% neutral buffered formalin, respectively, for the histological processing.The remaining samples were kept at -80 °C for RNA isolation and western blotting.

Biochemical assays Determination of serum thyroid hormones concentration
Total thyroxine (TT4), total triiodothyronine (TT3) and TSH concentrations of all groups were determined in the serum using ELISA kits acquired from Cal biotech INC (CBI, USA Company) according to the manufacturers' instructions and following the method of Thakur et al. [31], Maes et al. [32], and Burger and Patel [33] respectively.This assay uses the competitive inhibition enzyme immunoassay technique and the optical density was measured spectrophotometrically at 450 nm using Hitachi spectrophotometry (Tokyo, Japan).

Determination of serum testis hormones concentration
Luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone levels were measured in the blood serum by enzyme immunoassay kits (ALPCO Diagnostics) following the manufacturer's instructions.

Determination of kidney function biomarkers
Serum urea and creatinine were assayed by Spinreact commercial kits (Spain) depending on the methods of Coulombe and Favreau [34] and Larsen [35] respectively.

Determination of pro-inflammatory cytokines
TNF-α and IL-6 were estimated in testicular and renal tissue homogenates using R&D systems ELISA kits (USA) following the manufacturer's guidelines.

Determination of oxidative stress and antioxidant markers
In the homogenate of the testis and kidney, lipid peroxidation was assayed as malondialdehyde (MDA) level according to Preuss et al. [36].Also, reduced glutathione (GSH), glutathione peroxidase (GPx) and glutathione-s-transferase (GST) were determined following the methods of Beutler et al. [37], Matkovics et al. [38] and Mannervik and Guthenberg [39] respectively.

Histological examination
Twenty-four hours fixed renal and testicular tissues were washed with distilled water, dehydrated in an ascending ethanol series and paraffin wax embedded.For light microscopy inspection, paraffin sections were cut at a thickness of 4-5 μm via a rotary microtome and were stained with hematoxylin and eosin (H&E) [40].In each group, six sections were examined.At least eight microscopic fields per each section were scanned.Histopathological lesions of testis and kidney were examined and evaluated with a score formerly described as follows: (̶ ) no influence, (+) mild (< 25% of all fields showed alterations), (++) moderate (< 50% of all fields showed alterations) or (+++) severe changes (< 75% of all fields showed alterations) [41].

Gene expression analysis by qRT-PCR
Renal and testicular samples were treated with a TRIzol Plus RNA Purification Kit.The purity and concentration of RNA were detected spectrophotometrically. RNAs with A 260/280 ≥ 1.8 were chosen for cDNA synthesis.Up to 2 µg RNA was converted to cDNA by High-Capacity cDNA Reverse Transcription Kit. cDNA was amplified by Fast SYBR™ Green Master Mix using a set of two PCR primers that flank the target region as shown in Table 1.All kits were purchased from ThermoFisher Scientific, USA.RT-PCR conditions were set to 95 °C for 3 min, followed by 35 cycles involving denaturation (95 °C for 15 s), annealing (60 °C for 30 s) and extension (72 °C for 30 s).

Protein expression analysis by Western blotting
Frozen specimens at -80 °C were homogenized in an appropriate lysis buffer (RIPA) containing proteinase inhibitors, then centrifuged for 10 min to assay the protein concentration in the clear supernatant by Bradford reagent (ThermoFisher Scientific, USA).Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes, and blocked with a blocking buffer (5% skimmed milk dissolved in TBS/Tween 20 (TBST)).The membranes were incubated with primary antibodies, against Nrf2, HO-1, PPARγ, and β-actin (Santa Cruz Biotechnology, USA) diluted in the blocking buffer.After washing with TBST, the membranes were probed with secondary antibodies then washed.The bands were visualized with an enhanced chemiluminescence reagent (Bio-Rad, USA), quantified using Image J and normalized to β-actin.

Statistical analysis
Data were analyzed by SPSS (v.20).Results were represented as mean ± standard deviation (SD), and one-way ANOVA test was used to compare all data statistically, followed by Tukey's test post hoc analysis.A P value < 0.05 was stated significant.

Effect of ELT and HSP on circulating THs in CBZ-induced rats
The data recorded in Table 2 showed that CBZ-induced rats had a significant (P < 0.001) decrease in serum levels of TT3 and TT4 and a significant (P < 0.001) increase in serum level of TSH when compared with the control group.Treatment of hypothyroid rats with ELT or HSP showed significantly (P < 0.001) ameliorated serum levels of TT3, TT4, and TSH compared to CBZ-treated group.

Effect of ELT and HSP on FSH, LH, and testosterone hormones in CBZ-induced rats
The concentration levels of FSH, LH, and testosterone were significantly (P < 0.001) decreased in hypothyroid rats when compared with the control group.Treatment of hypothyroid rats with ELT significantly increased FSH (P < 0.01), LH (P < 0.001), and testosterone (P < 0.001) levels.Similarly, treatment of hypothyroid rats with HSP significantly (P < 0.001) increased FSH, LH, and testosterone levels as illustrated in Table 3.

Effect of ELT and HSP on renal function biomarkers in CBZinduced rats
As shown in Table 4, the levels of serum urea and creatinine were significantly elevated (p < 0.001) in CBZinduced group compared with the control group.Co-administration of ELT or HSP with CBZ significantly (p < 0.001) lowered serum levels of urea and creatinine comparing with the hypothyroid group.

Effect of ELT and HSP on pro-inflammatory cytokine production in testis of CBZ-induced rats
To investigate the protection by ELT and HSP against HPO-associated inflammation, tissue levels of the proinflammatory cytokines (TNF-α and IL-6) were determined.The levels of TNF-α (Fig. 1A) and IL-6 (Fig. 1B) in hypothyroid rats showed a significant (P < 0.001) increase when compared with the corresponding control group.Oral supplementation of either ELT or HSP with CBZ produced a significant (P < 0.001) decrease in the proinflammatory cytokines as shown in Fig. 1.

Effect of ELT and HSP on pro-inflammatory cytokine production in kidney of CBZ-induced rats
TNF-α (Fig. 2A) and IL-6 (Fig. 2B) were significantly (P < 0.001) increased in the renal tissues of hypothyroid rats compared to the control rats.Treatment of CBZinduced rats with ELT or HSP markedly (P < 0.001) decreased both TNF-α and IL-6 comparing with the hypothyroid rats as shown in Fig. 2.

Effect of ELT and HSP on testicular oxidant/antioxidant status in CBZ-induced rats
Hypothyroid rats showed a significant (P < 0.001) elevation in lipid peroxidation level when compared with the Fig. 2 Effect of eltroxin and hesperidin on pro-inflammatory cytokine production in kidney of CBZ-induced rats Fig. 1 Effect of eltroxin and hesperidin on pro-inflammatory cytokine production in testis of CBZ-induced rats controls (Table 5).Treating hypothyroid rats with ELT or HSP significantly (P < 0.001) reduced lipid peroxidation level.On the contrary, hypothyroid rats showed a marked (P < 0.001) decrease in testicular GSH content, GST and GPX activities when compared with the control rats.
Co-administration of ELT or HSP to hypothyroid rats significantly (P < 0.001) increased GSH, GST and GPX compared to CBZ-induced group (Table 5).

Effect of ELT and HSP on renal oxidant/antioxidant status in CBZ-induced rats
Lipid peroxidation level was elevated significantly (P < 0.001) in CBZ-induced rats compared to the control rats.Administration of ELT or HSP with CBZ showed a noticeable (P < 0.001) modulation in lipid peroxidation level as shown in Table 6.In contrast, GSH, GST, and GPX were significantly (P < 0.001) decreased in CBZ-induced rats compared to the control rats, while co-administration of ELT or HSP with CBZ showed a significant increase in GSH (p < 0.01), GST (p < 0.001) and GPX (p < 0.001) (Table 6).

Effect of ELT and HSP on gene and protein expression levels of Nrf2, HO-1 and PPARγ in the testis of CBZ-induced rats
We examined the efficacy of Nrf2/HO-1 signaling and PPARγ in defining the molecular mechanism controlling the protective impact of ELT and HSP on CBZ-induced hypothyroidism in rats.Nrf2 (Fig. 3A), HO-1 (Fig. 3B) and PPARγ (Fig. 3C) mRNA abundance in the testis of CBZ-induced group showed a significant (P < 0.001) down-regulation when compared with the corresponding control group.Oral treatment of the hypothyroid rats with either ELT or HSP produced a marked (P < 0.001) up-regulation of Nrf2, HO-1 and PPARγ mRNA expression in the testis (Fig. 3).
Western blotting data showed a significant (P < 0.001) decrease in the protein expression level of Nrf2 (Fig. 4A), HO-1 (Fig. 4B) and PPARγ (Fig. 4C) in the testes of CBZinduced rats when compared to the control rats.Oral supplementation of either ELT or HSP produced a significant (P < 0.001) increase in the protein expression level of Nrf2, HO-1 and PPARγ in the testes of the hypothyroid rats (Fig. 4).

Effect of ELT and HSP on gene and protein expression levels of Nrf2, HO-1, and PPARγ in the kidney of CBZinduced rats
Nrf2 (Fig. 5A), HO-1 (Fig. 5B), and PPARγ (Fig. 5C) mRNA abundance in the kidney of hypothyroid rats showed a significant (P < 0.001) decrease when compared with the control rats.Similarly, protein expression levels of Nrf2 (Fig. 6A), HO-1 (Fig. 6B), and PPARγ (Fig. 6C) were significantly (P < 0.001) down-regulated in the kidney of hypothyroid rats.Treatment of the hypothyroid rats with ELT or HSP produced a significant (P < 0.001) up-regulation of Nrf2, HO-1, and PPARγ mRNA and protein expression levels as shown in Figs. 5 and 6.

Effect of ELT and HSP on histopathological changes in the testis of CBZ-induced rats
H&E-stained sections of the testis from all experimental groups were represented in Fig. 7 and the detailed histopathological scores were summarized in Table 7.The control group revealed normal histology of seminiferous tubules which were oval or rounded with active spermatogenesis and lumen filled with frequent spermatozoa.Hypothyroid rats showed variable histopathological lesions in the form of severe irregular outlines of seminiferous tubules accompanied with degenerated germinal cells, deficient spermatogenesis, spermatogenic arrest, and vacuolization.Moderate degenerative spermatozoa in the affected lumens and necrosis of seminiferous tubules were also noticed.Severe empty seminiferous tubules could also be detected.In contrast, hypothyroid rats treated with ELT or HSP showed improvement in the testis sections with all mild pathological lesions.No influence of vacuoles in germ cells was observed in the hypothyroid rats treated with HSP.

Effect of ELT and HSP on histopathological changes in the kidney of CBZ-induced rats
Figure 8 showed the photomicrographs of H&E stained kidney sections from all experimental groups by light microscopic examination, while the detailed histopathological scores were illustrated in Table 8.The control rats showed the normal structure of both glomeruli and renal tubules.Rats received CBZ showed multiple histopathological changes, including severe glomerular degeneration and tubular necrosis, moderate dilation of the Bowman capsular space, vacuolar degeneration of renal tubules, together with blood vessels congestion.Concomitant treatment of the hypothyroid rats with ELT showed glomeruli and renal tubules improvement with mild glomerular degeneration and tubular necrosis.Hypothyroid rats treated with HSP showed improvement in pathological lesions with mild degenerative changes of glomeruli.

Discussion
HPO is a common endocrine disorder caused by a decline in the THs released from the thyroid gland.In the present study, the induction of HPO was evidenced by the significantly decreased T3 and T4 and the significantly increased TSH relative to the control group.These outcomes were parallel with the result of Farag et al. [43].The synthesis and release of TSH is negatively influenced by free T4 and T3 (unbounded to serum proteins) for keeping the levels of circulating THs within the normal range so when TH levels decrease, TSH secretion increases [44].ELT or levothyroxine is the synthetic form of T4 hormone and is used to treat HPO.It is identical to the naturally produced thyroid hormone in human [45].It has been proved in our work that ELT restored the changes in the levels of THs in harmony with the study of Abdel-Wahhab et al. [46].ELT can increase T4 and T3 secretion by up-regulating TSH receptors [47], or by increasing Fig. 3 Effect of eltroxin and hesperidin on gene expression level of Nrf2, HO-1 and PPARγ in the testis of CBZ-induced rats serum protein level through the regulation of DNA transcription and protein synthesis; since the circulating T4 and T3 are protein bound hormones [48].
A significant modulation in the hormonal profile of the thyroid gland was also achieved in CBZ-induced rats after treatment with HSP.HSP has several positive pharmacological effects including anti-inflammatory, anticarcinogenic, antiviral, hypolipidemic, and hypoglycemic without any recorded negative side effects [49,50].Our present results were confirmed by Hegazy et al. [51] who reported that hypothyroid rats treated with HSP showed an ameliorative effect on THs levels.This positive effect could rely on the modulatory effects of flavanones on thyroid and pituitary functions [52], or the ameliorative effect of HSP on the histopathological and ultrastructural changes induced in the thyroid gland during the hypothyroidism state [53].
In accordance with Mohebbati et al. [54], the present findings demonstrated that CBZ-induced HPO is associated with nephrotoxicity as revealed by the increase in serum urea and creatinine levels.Urea and creatinine concentrations increase during kidney function failure [55].On the contrary, co-treatment with ELT or HSP normalized urea and creatinine levels intensifying their renoprotective effect.According to Mooraki et al. [56], levothyroxine can reverse the decline in renal function caused by HPO.Additionally, in hypothyroid individuals with ischemic nephropathy, THs therapy quickly improved renal structure and function [57].Hence, ELT effect may be exerted through the normalization of the THs levels.Similarly, it was previously recorded that HSP reduced the rise in serum urea and creatinine levels in rats [58] and mice [59] with nephrtotoxicity.The renoprotective effect of HSP may be explained based on Fig. 4 Effect of eltroxin and hesperidin on protein expression level of Nrf2, HO-1 and PPARγ in the testis of CBZ-induced rats its antioxidant property, enhancement of cellular antioxidant defenses and free radical scavenging ability [60].
In accordance with the results of Amra et al. [61], our study showed that testosterone, FSH and LH levels were decreased in hypothyroid rats.Furthermore, Mohamed and Bushra [62] suggested that the anti-thyroid drugs may reduce the liver's capability to produce sex hormonebinding globulin (SHBG), which increases free androgen levels and inhibits the anterior pituitary's ability to release gonadotropin-releasing hormone (GnRH), resulting in decreased FSH and LH levels.On the opposite hand, treatment with ELT or HSP showed a significant increase in testosterone, LH and FSH levels.The protective effect of HSP against testicular toxicity induced by drugs and chemicals was recorded earlier [63,64].The modulation of testicular hormonal levels may be accredited to the recovery of THs levels by ELT and HSP treatment.T3 could increase LH receptors and steroidogenesis of leydig cells [65].T3 also stimulates testosterone secretion as a result of the activating action on leydig cells [66].Similarly, T4 amplifies SHBG concentration, peripheral aromatization of androstenedione, and testosterone levels [67].
The HPO condition is associated with elevated proinflammatory cytokines as illustrated by high levels of TNF-α and IL-6 in renal and testicular tissues after CBZ induction.Raised pro-inflammatory marker levels have been reported in some previous investigations in patients with HPO [68,69], and in animals′ renal tissue during a thyroid dysfunction state [70].Gupta et al. [71] also indicated a significant increase in IL-6 levels in hypothyroid patients compared to controls and this increase was positively correlated with serum TSH levels.Conversely, a negative correlation was found between IL-6 and serum TT3 or TT4 levels [72].
Treatment with ELT significantly decreased IL-6 and TNF-α levels, demonstrating the importance of early treatment in the abrogation of inflammation.These Fig. 5 Effect of eltroxin and hesperidin on gene expression level of Nrf2, HO-1, and PPARγ in the kidney of CBZ-induced rats findings were strengthened by Tayde et al. [73] and Goyal et al. [74] who reported a significant reduction in IL-6 and TNF-α levels in hypothyroid cases after treatment with levothyroxine.In contrary, Díez et al. [75] showed that increased TNF-α levels in hypothyroid patients did not return to normal following the restoration of thyroid function.Similarly, treatment of CBZ-induced rats with HSP markedly decreased renal and testicular IL-6 and TNF-α levels, indicating mitigated inflammation.Previous studies showed the anti-inflammatory effect of HSP in renal tissues [16,76] and testicular tissues [77].Under HPO conditions, HSP has been indicated to decrease inflammation in liver and lung as previously reported [51,78].
THs can modulate oxidative metabolism and have a considerable impact on OS and the antioxidant system [79].A positive correlation between lipid peroxidation and TSH was found; excess TSH directly produces OS [80].Therefore, the oxidant/antioxidant status was necessarily investigated.Treating rats with CBZ in the current study showed a significant increase in lipid peroxidation level and a significant decrease in GSH content and the activities of the antioxidant enzymes (GST and GPX), indicating an OS in testis and kidney.Similar to our findings, several studies indicated that HPO induced OS in testis and kidney [81,82].CBZ can disturb the balance between ROS generation and the antioxidant defense systems [78].This is consistent with Wang et al. [83] who believed that cell damage and testicular dysfunction observed during HPO resulted from the buildup of free radicals and increased lipid peroxidation and OS.Similarly, the kidney is susceptible to damage triggered by Fig. 6 Effect of eltroxin and hesperidin protein expression level of Nrf2, HO-1, and PPARγ in the kidney of CBZ-induced rats ROS because renal lipids contain a plenty of polyunsaturated fatty acids [84].
The present data showed that treatment with ELT and HSP reduced lipid peroxidation and improved enzymatic and non-enzymatic antioxidant defenses evidencing the ability of preventing oxidative stress as a mechanism to counteract HPO related renal and testicular damage.Chakrabarti et al. [85] showed that treatment of HPO with levothyroxine reduced the stress markers to a significant degree.THs have a critical role in minimizing oxidative stress by promoting the expression of antioxidant enzymes and acting as radical scavengers [86].As well, HSP showed an antioxidant capacity in animal models of testicular [87,88], hepato-renal [89,90], and renal [12] toxicity.
To inspect the protective mechanism of ELT and HSP, we examined their effect on Nrf2/HO-1 signaling pathway.In the current work, hypothyroid rats showed a declined mRNA and protein expression of Nrf2 and HO-1, due to exaggerated generation of ROS.Several experiments demonstrated decreased Nrf2/ARE/HO-1 in situations with high ROS generation [91,92].Downregulation in Nrf2/HO-1 pathway was illuminated not only in testicular toxicity [93], but also in nephrotoxicity [94,95] caused by toxic agents.On the opposite side, Nrf2 and HO-1 were significantly up-regulated in the hypothyroid group treated with HSP.Our results confirmed prior studies where HSP showed a cyto-protective effect by up-regulating Nrf2/HO-1 pathway in testicular toxicity [96] and nephrotoxicity [16,97].In the same trend, treating hypothyroid rats with ELT significantly increased Nrf2/HO-1 signaling.This result was confirmed by the increased expression of Nrf2 and HO-1 in the liver [51] and lung tissues [78] of hypothyroid rats treated with ELT.
Gene and protein expression of PPARγ was significantly decreased in both testis and kidney of hypothyroid rats.The inhibition of PPARγ expression was previously reported in testicular damage induced by toxic substances [98].Inan et al. [99] also reported that testicular OS in rats can be increased following PPARγ inhibition.Additionally, there is a proof that PPARγ plays an anti-inflammatory effect in inflammatory disorders [100].Increased expression of TNF-α and interleukin-1 beta (IL-1β) was suggested as the mechanism of PPARγ down-regulation in testicular tissues [101].This ensures the negative correlation between PPARγ activation and TNF-α reduction that was documented in our study.Furthermore, increased OS and pro-inflammatory cytokines were reported to down-regulate PPARγ expression during renal failure [102].Our findings reported that ELT or HSP exert beneficial effects on the testis and kidney through PPARγ activation.Several research studies reported that PPARγ agonists provided protection against HPO-induced toxicity in many organs [103][104][105].Other reports suggested that PPARγ activation protected against renal failure models such as chronic renal allograft damage [106] and ischemia-reperfusion renal injury [107].In addition, the antioxidant and anti-inflammatory properties of HSP against cyclophosphamide-induced hepatotoxicity [108] and isoproternol-induced cardiotoxicity [109] were performed through up-regulating PPARγ.
As a result, coactivation of Nrf2 and PPARγ appears to be the mediator of the antioxidant and anti-inflammatory effect of HSP and ELT.This can be explained since PPARγ response element is present in the Nrf2 gene's promoter region [110].Also, it has been shown that Nrf2 activates PPARγ and guards against oxidative damage [111].
Our histopathological findings were consistent with the biochemical results intensifying the results of Amra et al. [61] and Alhealy et al. [82] who clarified the testicular changes induced by HPO.Adverse effects in histological structure and semen parameters in adult male hypothyroid rats were also reported by Ibrahim et al. [112].Coadministration of ELT or HSP showed improvement in testicular structure as previously reported [61,87].Their protective effect on HPO-induced testicular degeneration may be attributed to the antioxidant abilities, which eliminate free radicals to minimize lipid peroxidation and therefore preserve normal histological structure of testes [77,113].
In the present study, hypothyroid rats showed histopathological changes in the renal tissues including damage in the glomeruli and tubules as were previously notified by Ubaid et al. [114].Treatment of hypothyroid rats with ELT attenuated the severity of renal injuries except mild glomerular degeneration and tubular necrosis, while hypothyroid rats treated with HSP revealed improvement in renal tissues but some degenerative changes of glomeruli were still visible.The improvement in kidney structure provided further evidence of the antioxidant properties of ELT and HSP.Lashein et al. [115] reported that co-administration of levothyroxine with CBZ ameliorated renal histological changes.Similarly, Küçükler et al. [90] indicated that the antioxidant capacity of HSP was responsible for the protective efficacy against renal toxicity induced in rats by chlorpyrifos.

Conclusions
This investigation proved that administration of ELT and HSP ameliorated THs concentration, testis hormonal profile, kidney function biomarkers, OS, and inflammation in rats with CBZ-induced HPO.These ameliorating effects were achieved through up-regulation of PPARγ and Nrf2/HO-1 signaling pathway.Histological changes of the testes and kidneys were also improved.Therefore, HSP could be a promising element in the development of a potent antihypothyroid agent.Also, we encourage patients with HPO to consume HSP rich foods along with medication to protect their body organs from OS and inflammation.

Table 1
Primer sequences used for qRT-PCR

Table 2
Effect of ELT and HSP on serum TT3, TT4, and TSH hormones levels of hypothyroid rats

Table 3
Effect of ELT and HSP on serum FSH, LH, and testosterone hormones levels of hypothyroid rats ***###

Table 4
Effect of ELT and HSP on serum urea and creatinine of hypothyroid rats

Table 5
Effect of ELT and HSP on testicular oxidant/antioxidant status of hypothyroid rats

Table 6
Effect of ELT and HSP on renal oxidant/antioxidant status of hypothyroid rats

Table 7
Grading of histopathological alterations obtained from testicular tissues of different experimental groups Effect of eltroxin and hesperidin on histopathological changes in the testis of CBZ-induced rats

Table 8
Grading of histopathological alterations obtained from renal tissues of different experimental groups Fig.8Effect of eltroxin and hesperidin on histopathological changes in the kidney of CBZ-induced rats